Anti-Fungal Composition

ABSTRACT

The present invention relates to an improved anti-fungal composition, to a process for preparing it and to its use as a preservative.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. application Ser.No. 13/453,833, filed Apr. 23, 2012, which is a Continuation applicationof U.S. application Ser. No. 12/067,208 filed Jun. 3, 2008, now U.S.Pat. No. 8,187,844, issued on May 29, 2012, which is a National StageApplication of PCT/EP2006/066909, filed Sep. 29, 2006, which claimspriority to European Patent Application No. 05109190.8 filed Oct. 4,2005, the content of all of which are hereby incorporated by referencein their entireties.

BACKGROUND

1. Field of the Invention

The present invention relates to an improved anti-fungal composition, toa process for preparing such an anti-fungal composition and to its use.

2. Description of Related Art

The need for improved food preservation methods is great. It has beenestimated that about one quarter of the world's food supply is lost as aresult of microbial spoilage and food-borne microbial infectionsrepresent a constant and serious threat to human health.

Fungal spoilage can lead to serious economic losses. Several foodproducts e.g. agricultural products, dairy and meat products, fruits andvegetables and derived products, bakery products and cosmetics are verysusceptible to fungal growth. Examples of dairy products are cheese,cottage cheese, ricotta and yoghurt. Dried cured sausages are an exampleof meat products. Examples of agricultural products are crops such ascereals, nuts, fruits, vegetables and flower bulbs. Spoilage by fungidoes not only affect the quality of the product, but also represents ahealth risk. It is well known that some fungal species, which grow one.g. dairy products and sausages, can produce mycotoxins. Somemycotoxins are extremely dangerous as they can cause lethal diseases.Therefore the outgrowth of unwanted fungi in and on food products shouldalways be prevented.

Food preservation techniques, e.g. heat processing, freezing,ultrasound, irradiation, and modified atmosphere packaging,significantly reduce microbial load but of particular concern is theevidence that processed foods are being contaminated with microorganismsfollowing processing and prior to packaging. Of rising concern in thefood industry is microbial spoilage of various foods such as dairy andmeat products, dressings, spreads, margarines and seafood. Especiallyfood products in the 2.0 to 7.0 pH range are known to be susceptible tomicrobial spoilage by yeast, fungi, acid tolerant bacteria and/ormesophilic or thermophilic spore forming and non-spore forming bacteria.

Mostly, processed foods are not eaten directly after processing therebypermitting bacteria, yeast or mould introduced by post-contamination togrow. Since food consumption may occur without reheating the processedfoods to sufficient temperatures for sufficient time, there is a risk offood poisoning or food spoilage. Furthermore, the recent trend forminimally processed foods with the intrinsic nutritional and sensoryqualities of raw and fresh foods has raised a new safety risk. Milderpreservation treatments, such as high hydrostatic pressure and pulsedelectric fields have proved to be successful but rely on effectivehurdles i.e. cold chain and addition of natural anti-microbials.

There has been extensive research conducted in the field of food safetyto develop effective anti-fungal compositions. Natamycin also known aspimaricin or tennecetin, is a polyene antibiotic, which has been knownsince the late fifties (Struyk et al, Antibiot. Ann. 1957-1958, 878) andwhich is currently used as a preservative in many food and agriculturalproducts. U.S. Pat. No. 5,821,233 discloses natamycin exhibiting a highrelease rate of at least 3 μg/24 hours over the first 24 hours whencontacted on a carrier with an agar surface of 0.6 cm diameter, and acarrier loading of 40 μg of natamycin. U.S. Pat. No. 5,997,926 disclosesnatamycin complexes with similar release rates. For some foodapplications, this kind of natamycin with a high release rate does notoffer an adequate protection because for example the duration of theprotection is not sufficient and/or the stability of the natamycin isnot optimal. As a consequence, higher amount of natamycin would beneeded to offer an adequate protection. Natamycin with a high releaserate can also penetrate too far in the product treated, which could beunwanted in some products.

To control the release of natamycin, encapsulation has been proposed,see for example WO2005/018322 and US2005/042341. Encapsulation does notchange the characteristics of the natamycin itself, but it tries toensure a slower release of natamycin by applying an extra barrier. Thisautomatically requires extra formulation steps after the recovery of thenatamycin from a natamycin source, wich makes the natamycin productionprocess more complicated then when the natamycin itself would be changedto give a slower release.

Therefore, there is still a need for improved anti-fungal compositionsthat could solve at least some of these problems: slower release, longerduration of protection and/or improved stability.

SUMMARY

The present invention relates to an improved anti-fungal composition,especially a natamycin is provided, which exhibits a low natamycinrelease rate. This natamycin is preferably prepared using the processdefined below.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT Process for PreparingNatamycin Exhibiting a Low Natamycin Release Rate

The present invention relates to a process for the production of thenatamycin of the invention with a low natamycin release rate as definedbelow. In the process of the invention, natamycin is recovered from afermentation broth containing biomass and natamycin and said processcomprises:

-   -   (a) disintegrating the biomass;    -   (b) separating the natamycin from the thus treated fermentation        broth to obtain a natamycin suspension;    -   (c) adjusting the pH of said natamycin suspension to a value        greater than 10 and adding an amount of a substantially        water-miscible solvent sufficient to dissolve the natamycin in        said natamycin suspension;    -   (d) removing insoluble solids from said pH-adjusted natamycin        solution;    -   (e) lowering the pH of said solution obtained in step (d) to a        level sufficient to precipitate the natamycin and to form a        natamycin suspension; and    -   (f) removing the natamycin from said natamycin suspension.    -   (g) Optionally, drying of the said removed natamycin    -   (h) And optionally reducing the size of the said dried        natamycin.

The first advantage of the process according to the invention is that,without encapsulation or other way of complex processing, it leads to anatamycin product which has a slower release rate than high releasenatamycin formulations, such as U.S. Pat. No. 5,997,926 and U.S. Pat.No. 5,821,233, and other commercially available natamycin products, suchas DELVOCID® and DELVOCID® Instant, as illustrated in the Examples. Atthe same time, the release rate is high enough to offer longerprotection. Surprisingly, the production process according to theinvention leads to a product with new characteristics which could nothave been anticipated from the prior art.

A second advantage is that the process maintains the advantage ofearlier processes, see for example WO 97/29207, that natamycin isseparated from the biomass and other impurities without using organicsolvents, which is preferable from an environmental point of view.

Fermentations producing natamycin usually result in a fermentation brothcomprising natamycin, biomass solids, dissolved or suspended nutrients,other fermentation products and water.

The fermentation broth in general contains of at least 2 g/l natamycin,preferably at least 7 g/l natamycin. For example the natamycinconcentration in the fermentation broth can be about 7 g/l as disclosedin WO 93/03170. Since natamycin has a very low solubility in water undertypical fermentation conditions, the natamycin in the fermentation brothis present in solid form. Preferably, the natamycin is mainly present insolid form. Mainly means at least 50%, preferably at least 70% and morepreferably at least 80%. Solid natamycin means ‘natamycin not dissolvedin water’. The solid form of natamycin present in the fermentation brothmay preferably comprise natamycin particles. Natamycin particles arenatamycin crystals, which, for example, may have the following forms:needle-formed crystals, disc-formed crystals or the like. During thefermentation natamycin particles are formed. The natamycin particlesusually have diameters ranging from 0.5-20 micrometer. The diameter ofthe natamycin particle is the largest distance from one part of theparticle to the other end of the particle. Needle-formed natamycinparticles with diameters of more than 40 micrometer have been observed.Diameters may be determined using a microscope. Preferably, in theprocess of the invention, the fermentation broth comprises natamycinparticles having an average particle diameter of at least 2 micrometer,more preferably the natamycin particles have an average particlediameter of at least 5 micrometer and most preferably the natamycinparticles have an average particle diameter of at least 10 micrometer.

The biomass of the Streptomyces organisms used in the production ofnatamycin generally consists of clusters (mycelia) of threads, althoughother forms of biomass, e.g. the so-called “pellets”, may be present aswell. In these threads (hyphae) compartments are present, in whichcellular activities are localized. The size of these threads as presentin the clusters is in general from 10-30 micrometer (diameters rangingfrom 0.5-1.0 micrometer).

According to a preferred process, the natamycin is mainly present insolid form in the fermentation broth. Preferably, at least 50% of thenatamycin is present in solid form, more preferably at least 70%, evenmore preferably at least 80%.

Step (a)

In the present invention, the fermentation broth obtained at the end ofthe fermentation process is treated to disintegrate the biomass.Disintegration of the biomass may result in lysis, solubilisation ofcell matters, and fragmentation (size reduction) of the clusters andthreads. Disintegration of the biomass may be checked by viewing thebiomass with a microscope (magnification 400×). Disintegration iscomplete if hardly any clusters or threads of the biomass can beobserved through a microscope. Disintegration of the biomass can also bedetermined by measuring the viscosity of the fermentation broth. Forexample during the disintegration of a biomass of the cluster-type, theviscosity decreases. If the viscosity does not substantially decrease onfurther treatment, the biomass will be sufficiently disintegrated.Although different fermentation conditions or different Streptomycesorganisms used in the production of natamycin may result in somewhatdifferent forms of biomass present at the end of the fermentation, oneskilled in the art is able to find a suitable duration for thedisintegration of the biomass of any fermentation broth.

Homogenization, high shear mixing and ultrasonic techniques or heat-,pH-(alkaline), or enzymatic-treatments or treatment with surface-activeagents can, for example, be used alone or in combination to disintegratethe biomass. The disintegration techniques are chosen in such a way thatdisintegration is obtained without substantially affecting thenatamycin. Most of the natamycin, at least 80%, preferably up to 100%,keep their solid form and natamycin activity will not substantiallyreduce. Furthermore, it will be clear to one skilled in the art that thedisintegration techniques may not substantially affect the natamycinparticle size. If particle size of natamycin is reduced like theparticle size of the biomass, then separation of the natamycin from thebiomass would be difficult. An efficient example of disintegration isthe use of a heat treatment optionally combined with a pH-treatment.

A heat treatment can be applied to the fermentation broth at the end ofthe fermentation (e.g. in the fermentor, after all supplies (e.g.oxygen, carbon or nitrogen sources) have ceased). The heat treatment maybe carried out, for example, for 1 to 8 hours and, for example, at 30 to50° C. Preferably the heat treatment may be carried out at 30 to 40° C.Higher temperatures may result in flocculation, precipitation andcoagulation, which would adversely affect separation of the biomass fromthe natamycin particles.

A pH-treatment for, for example, 1 to 8 hours and, for example, at a pHof 8 to less than about 10 can also be easily conducted at the end ofthe fermentation in the fermentor. At pH's above 10 natamycin willbecome more soluble and more vulnerable to inactivation, which mightadversely affect recovery yield and purity of the final natamycin.Sodium hydroxide or any other compatible caustic material, for exampleammonium hydroxide or potassium hydroxide, can be used to increase thepH. After the alkaline incubation the broth is neutralized byhydrochloric acid or another compatible acid, for example phosphoricacid, sulphuric acid or acetic acid. Preferably, neutralization takesplace after separating the natamycin from the fermentation broth.

Enzymatic-treatments can involve the incubation with cell walldecomposing and/or organic polymer decomposing enzymes such as lysozyme,xylanase, cellulase, protease, glucanase, lipase and amylase. Theenzymes, alone or as mixtures of enzymes, are generally incubated underthe optimum conditions for the enzymes to operate. The enzymescontribute to the lysis of cells and to the solubilization of organicpolymers.

Homogenisation can involve the use of a Manton-Gaulin typehomogenisator. The fermentation broth is forced through an orifice. Dueto pressure forces the biomass will disintegrate.

Disintegration of the biomass by ultrasonic techniques can be obtainedby applying ultrasonic waves to the fermentation broth, that willprovide for oscillation of cell liquid which the cell walls cannotwithstand. Disintegration of the biomass by high shear mixing involvesthe application of high shear forces to the biomass. These high shearforces can be obtained by stirring or other mechanical agitation.Certain fermentors may for example be equipped with stirring deviceswhich are capable of providing the required high shear forces in orderto disintegrate the biomass. During fermentation, stirring can beadapted to the optimal growth or natamycin production conditions for thebiomass. After fermentation stirring can be adapted in order todisintegrate the biomass for example by applying high stirringvelocities. High shear mixing may also for example be accomplished byusing high shear Waring (or other) blenders.

Disintegration of the biomass by treatment with surface-active agentsmay involve for example the use of octylphenoxypolyethoxyethanolcompounds, such as Triton type compounds. The fermentation broth may beincubated with for example 0.01 to 1% of for example Triton X-100 duringfor example 1 to 24 hours.

Step (b)

After the biomass disintegration-step, the natamycin is separated fromthe biomass to obtain a natamycin suspension. Due to the disintegrationtreatment, the biomass now mainly consists of small solid particlesand/or solubilized matter. Where conventional separation techniques usedin the recovery processes for fermentation products are mainly used toseparate the solid from the liquid phase, the separation techniquespreferably used in the present invention separate solid particles fromthe disintegrated biomass, for example on the basis of size differencesand/or density differences. The separation techniques preferably used inthe present invention will not result in a clear liquid phase, but willresult in a troubled liquid phase that contains most of the smallerand/or less dense solid particles (mainly comprising the disintegratedbiomass).

In order to separate the biomass from the natamycin particles thefermentation broth can, for example, be treated using a gravity gradientseparation technique. The gravity gradient separation techniqueseparates the natamycin particles from both soluble and insolubleimpurities. Gravity gradient separation techniques include, for examplegravity gradient centrifugation, and may, for example, use upflowcolumns and hydrocyclones. Gravity gradient separation techniques makeuse of the principle that particles of different densities and/or sizescan be separated when these particles of different densities and/orsizes are subjected to gravity or equivalent forces.

During the biomass disintegration the biomass particles become smaller.This makes it possible to separate the biomass from the natamycinparticles. Usually more than 90% of the disintegrated biomass and otherimpurities can be removed with the gravity gradient separationtechnique. Separation efficiency can be increased by adding water and/ora salt (e.g. sodium chloride) to the disintegrated fermentation broth.

The use of gravity gradient separation techniques has the advantage thatit is possible to easily modify or direct the process according to thedesired purity and yield of the final product. By varying the operationconditions, the purity or the yield can be increased. In general whenthe purity increases, the yield will decrease and vice versa. Theprocess according to the invention can for example provide natamycin ofabout 70 w/w % purity (anhydrous basis) on dry matter with a yield ofabout 90%. Using different process parameters natamycin of about 90 w/w% purity (anhydrous basis) on dry matter with a yield of about 80% canalso be obtained with the process of the present invention. It is evenpossible to produce several products of different qualities from onefermentation broth.

Gravity gradient separation techniques will give better results, e.g.higher purities and/or yields, if the difference in particle densityand/or size between the product particles and the impurities isincreased. Therefore it is preferred that the fermentation brothcontains natamycin particles having an average particle diameter of atleast 2 micrometer. The diameter is preferably determined using amicroscope. Preferably the average natamycin particle diameter is atleast 5 micrometer, more preferably at least 10 micrometer. Fermentationbroths containing natamycin particles with an average diameter of about25 micrometer have been observed. Since natamycin may be present in thefermentation broth with diameters ranging from below 0.05 micrometer toabout 40 micrometer, it will be clear to one skilled in the art that thesmallest particles may be lost during separation. Furthermore, it willbe clear to one skilled in the art that fractions of natamycin particleswith large diameters of high purity may be obtained using the gravitygradient separation technique. The conditions under which the gravitygradient separation technique is operated determine which fraction ofthe natamycin will be recovered. In general larger natamycin particlescan, for example, be obtained by using low shear conditions during thefermentation, or by seeding the fermentation with small natamycinparticles or by prolonging the fermentation.

Gravity gradient centrifugation can be simulated on laboratory scale byoperating a batchwise centrifuge for a shorter time or with a lowernumber of revolutions per minute as compared to the standard operationof the centrifuge, which would result in a clear separation of thesolids from the liquids.

On a production scale the centrifuge is usually operated continuously.As compared to the standard operation of this type of centrifuge, thehold up time in the centrifuge is decreased in order to separate thenatamycin from the disintegrated biomass. The lower the hold up time,the higher the purity and the lower the yield of the obtained natamycin.One skilled in the art is able to find a suitable hold up timecorresponding to an optimized or desired purity:yield ratio.

At the end of step (b), the natamycin suspension obtained has preferablya total volume which is approximately 5 to 10% compared to the originalfermentation broth and which contains preferably less than 20% v/v ofrest of biomass. More preferably, the natamycin suspension obtained hasa total volume which is approximately 6 to 8% compared to the originalfermentation broth and which contains less than 15% v/v of rest ofbiomass. Most preferably, the natamycin suspension obtained has a totalvolume which is approximately 6 to 8% compared to the originalfermentation broth and which contains less than 10% v/v of rest ofbiomass.

Step (c)

In a following step of the process of the invention, the pH of saidnatamycin suspension is adjusted to a value of at least 10 and an amountof a substantially water-miscible solvent sufficient to dissolve thenatamycin in said natamycin suspension is added.

Preferably, the water-miscible solvent used in step (c) is selected fromthe group consisting of acetone, methanol, ethanol, propanol,isopropanol, propanediol, tetrahydrofuran.

According to a preferred embodiment, the pH of step (c) is adjusted to avalue ranged between 10.0 and 11.0, preferably between 10.2 and 10.8,more preferably between 10.3 and 10.6 and most preferably adjusted to avalue of about 10.4.

The pH of the natamycin suspension is preferably adjusted by adding anappropriate quantity of an alkaline agent such as NaOH, KOH, Na₂CO₃,K₂CO₃.

The addition of a water-miscible solvent and the increase of the pH ofthe natamycin suspension leads to a solution comprising dissolvednatamycin.

Step (d)

Subsequently, any insoluble solids are removed from said pH-adjustednatamycin solution. The removal of the insoluble solids may be performedusing any suitable method such as membrane filtration, depth filtrationor centrifugation. One skilled in the art will be able to perform theinsoluble solids removal using any of the techniques described above.Preferably, the removal of the remaining debris is made by membranefiltration followed by a depth filtration. After the membranefiltration, the filter cake is washed with a propanol solution to removeany dissolved natamycin from the cake. Preferably, the propanol solutionis 35% v/v. Preferably, the depth filtration is realized on a filterhaving at least 0.4 μm pore diameter. More preferably, the filter has atleast 0.25 μm pore diameter.

Step (e)

Subsequently, the pH of the natamycin solution is lowered to a levelsufficient to precipitate natamycin and to form a natamycin suspension.Any acidic chemical can be used to lower the pH. An example of asuitable acidic chemical is hydrochloric acid. The pH is lowered to avalue ranged between 5.0 and 8.0. Preferably the pH is lowered to avalue ranged between 5.0 and 7.0, more preferably between 5.5 and 6.5.Most preferably, the pH is lowered to reach a value of about 6.0.Preferably, the pH is lowered using small pH steps in order to ensurethe formation of crystalline crystals possessing high purity (preferablyunder continuous stirring).

A small pH step means preferably that the addition of a suitablequantity of an acidifying agent is added to reach a lowering of 0.1,preferably 0.2 and more preferably 0.3 unit of pH per 5 minutes. The pHis preferably lowered 0.3 unit of pH per 5 minutes until the solutionbecomes turbid. After stirring continuously at this pH, the pH islowered with 0.3 pH units per 3 minutes until the pH is 6.0.

Optionally, before lowering the pH of the natamycin solution, thetemperature of this solution may be increased from 20° C. to 35° C.

After crystallization the temperature of the natamycin slurry is loweredto 5° C. to increase the yield.

Step (f)

In a further step of the process of the invention, natamycin is removedfrom said natamycin suspension. Preferably, the natamycin is removedusing a membrane filter press. After filtration, the natamycin is washedusing a propanol solution and subsequently dried by aeration of thenatamycin filter cake. Preferably, the propanol solution is 35% v/v.Preferably, the produced filter cake is once more suspended using a 70%propanol solution and subsequently filtered to further reduce the watercontent.

Step (g)

Optionally, after the separation step the natamycin suspension may, forexample, be dried in order to obtain a dry product. Any convenientdrying technique can be used, e.g. vacuum drying, conduction drying orconvection drying. Since natamycin is stable in the crystalline formthereof [natamycin.3H₂O], it is critical not to dry the product to amoisture content below about 7%. Vacuum drying is preferably conductedat about 40° C.

Preferably, the drying is performed using a Nauta-mixer operated undervacuum. Drying is finished when a temperature of 40° C. is reached.Preferably, after drying, the temperature of the natamycin is reduced tobelow 20° C. to prevent microbial growth of any possible contamination.

Step (h)

Optionally, the dried natamycin obtained in step (g) is crushedpreferably using a hammer mill in order to obtain crystals having adiameter ranged between 1 to 50 μm, preferably 1 and 10 μm. According toa preferred embodiment, the natamycin is crushed as described in U.S.Pat. No. 6,576,617.

Natamycin

Surprisingly the natamycin obtainable by said process exhibits a lowrelease rate and/or a decrease in release rate with respect to natamycinknown in the art. Therefore, the invention also relates to a natamycinobtainable by said process described above. The natamycin according tothe invention exhibits a low release rate and/or a decrease in releaserate as defined below. According to a preferred embodiment, thenatamycin of the invention is not incorporated in any specificformulation to get these features. It is the natamycin per se thatexhibits these features. The skilled person will understand that thescope of the invention is not limited to natamycin prepared according tothe process of the invention.

Natamycin Defined by Having a Specific Decrease in Release Rate

According to a preferred embodiment, the release rate of the natamaycinof the invention decreases by 1 to 45 percent after at least 9 days,when contacted on a carrier with an agar surface of 0.6 cm diameter, anda carrier loading of 5 to 40 μg of natamycin. More preferably, therelease rate under these conditions decreases by 10 to 45 percent, evenmore preferably by 20 to 45 percent and most preferably by 40 to 45percent.

Preferably, the release rate of the natamycin decreases by 1 to 45percent after 9 days, when contacted on a carrier with an agar surfaceof 0.6 cm diameter, and a carrier loading of 40 μg of natamycin. Morepreferably, the release rate under these conditions decreases by 10 to45 percent, even more preferably by 20 to 45 percent and most preferablyby 40 to 45 percent after 9 days.

Even more preferably, the release rate of the natamycin decreases by 1to 35 percent after 9 days, when contacted on a carrier with an agarsurface of 0.6 cm diameter, and a carrier loading of 10 μg of natamycin.More preferably, the release rate decreases by 10 to 35 percent, morepreferably by 20 to 35 percent and most preferably by 30 to 35 percent.

Natamycin Defined by Having a Specific Release Rate

According to another preferred embodiment, natamycin exhibits a releaserate ranged between 6 and 10 percent of the initial carrier loadingafter at least 9 days, when tested on a carrier with an agar surface of0.6 cm diameter, and a carrier loading of 5 to 40 μg of natamycin. Morepreferably, the release rate ranges between 6 and 8 percent, morepreferably between 6 and 7 percent and most preferably between 6 and 6.5percent.

Preferably, natamycin exhibits a release rate ranging between 0.60 and0.90 μg/day after 11 days, when tested on a carrier with an agar surfaceof 0.6 cm diameter, and a carrier loading of 10 μg of natamycin. Morepreferably, the release rate ranges between 0.60 and 0.80, morepreferably between 0.60 and 0.70 and most preferably between 0.60 and0.65.

Even more preferably, natamycin exhibits a release rate ranging between0.35 and 0.50 μg/day after 11 days, when tested on a carrier with anagar surface of 0.6 cm diameter, and a carrier loading of 5 μg ofnatamycin. More preferably, the release rate ranges between 0.35 and0.45 and most preferably between 0.35 and 0.40.

According to a preferred embodiment, natamycin exhibits:

-   -   a release rate which decreases by 1 to 45 percent after at least        9 days, when contacted on a carrier with an agar surface of 0.6        cm diameter, and a carrier loading of 5 to 40 μg of natamycin        and/or,    -   a release rate ranging between 6 and 10 percent of the initial        carrier loading after at least 9 days, when tested on a carrier        with an agar surface of 0.6 cm diameter, and a carrier loading        of 5 to 40 μg of natamycin, and    -   a release rate ranging between 0.1 and 2.0 μg per 24 hours over        the first 24 hours, when contacted on a carrier with an agar        surface of 0.6 cm diameter, and a carrier loading of 40 μg of        natamycin. More preferably, the release rate is ranging between        0.5 and 2.0, more preferably between 1.0 and 2.0 and most        preferably between 1.5 and 2.0    -   optionally, natamycin crystals have a diameter which ranges        between 1 to 50 μm, preferably 1 and 10 μm.

According to a preferred embodiment, natamycin exhibits:

-   -   a release rate which decreases by 1 and 45 percent after at        least 9 days, when contacted on a carrier with an agar surface        of 0.6 cm diameter, and a carrier loading of 5 to 40 μg of        natamycin and/or,    -   a release rate ranging between 6 and 10 percent of the initial        carrier loading after at least 9 days, when tested on a carrier        with an agar surface of 0.6 cm diameter, and a carrier loading        of 5 to 40 μg of natamycin, and    -   a release rate ranging between 0.1 and 1.0 μg per 24 hours over        the first 24 hours, when contacted on a carrier with an agar        surface of 0.6 cm diameter, and a carrier loading of 10 μg of        natamycin. More preferably, the release rate ranges between 0.2        and 1.0, more preferably between 0.4 and 1.0 and most preferably        between 0.8 and 1.0.    -   optionally, natamycin crystals have a diameter which ranges        between 1 to 50 μm, preferably 1 and 10 μm.

The test used to measure the natamycin release rate has been describedin example 1 of this application. Briefly, the sample comprisingnatamycin is applied to a carrier. The carrier can be any material thatprovides unlimited water transport. Preferably, the carrier is a filterpaper disc, more preferably the filter paper disc used in example 1.Subsequently, the carrier loaded with the natamycin composition isapplied to the surface of an agar plate seeded with yeast or fungalcells (in example 1 Saccharomyces cerevisiae cells were used). The agarplate and the carrier are subsequently incubated at a temperature suchthat the natamycin is released into the agar. (in example 1, thetemperature chosen is 6° C.) during 24 hours. After having removed thecarrier from the agar plate, the agar plate is incubated under growthpermitting conditions for said cells (in example 1, the temperature is30° C.) during a chosen period. In this case, the period is 24 hours or48 hours depending on the type of release rate desired to be determined.Finally, inhibition of cell growth in the agar due to the presence ofthe natamycin, which has been released from the carrier into the agar,is determined. The size of the inhibition zone is a measure of thenatamycin released from the sample.

Natamycin with such a low natamycin release rate is very attractive forall applications wherein for example a long protection is desired. Forexample, in cheese or in sausages or in other meat products such aspoultry or in seafood and bakery products wherein a protection of atleast several weeks against moulds is desired.

According to a preferred embodiment, a water-miscible solvent is presentwith the natamycin of the invention. Preferably, the water-misciblesolvent is selected from the group consisting of acetone, methanol,ethanol, propanol, propanediol, tetrahydrofuran and combinationsthereof.

Use of the Natamycin of the Invention

The invention further relates to an anti-fungal composition comprisingthe natamycin of the invention exhibiting a low natamycin release rate.According to a preferred embodiment, the anti-fungal composition of theinvention additionally comprises at least another anti-microbial agentselected from the group consisting of a weak acid preservative, sulphurdioxide, sulphite, nitrate, nitrite, dimethyl dicarbonate, biphenyl,diphenyl, orthophenylphenol, thiobendazole, an inorganic acid, animidazole and a bacteriocin. All these components are already known tothe person skilled in the art and briefly described below:

-   -   1. a weak acid preservative such as sorbic acid, propionic acid,        benzoic acid, a p-hydroxybenzoic acids, lactic acid, citric        acid, acetic acid or an alkali metal or alkali earth metal salt        thereof;    -   2. a polyene anti-fungal compound, preferably natamycin;    -   3. sulphur dioxide or sulphites;    -   4. nitrate and nitrite;    -   5. dimethyl dicarbonate;    -   6. biphenyl, diphenyl, orthophenylphenol or thiobendazole;    -   7. an inorganic acid, such as hydrochloric acid;    -   8. an imidazole such as imazalil; and/or    -   9. any anti-fungal compound known in the art for use as a        preservative for food products, crop protection or after-harvest        treatment of fruits, vegetables orcereals, pharmaceutical or        cosmetic products.    -   10. nisin or pediocin or lysozyme.

Preferably the anti-microbial agent is a weak organic acid preservativeand/or natamycine. The weak organic acid preservative may be sorbicacid, propionic acid, benzoic acid, lactic acid, citric acid or analkali metal or alkali earth metal salt thereof, or mixtures thereof.According to a more preferred embodiment, the anti-microbial agent issorbic acid, potassium, or calcium sorbate; benzoic acid, sodium,potassium, or calcium benzoate; natamycine or mixtures thereof.

Anti-microbial composition comprising a bacteriocin will be activeagainst bacteria. Nisin is a peptide-like antibacterial substanceproduced by microorganisms such as Lactococcus lactis subsp. lactis. Itis mainly active against gram-positive bacteria. Nisin is non-toxic andis free of side effects. Nisin is a Generally Recognized as Safe (GRAS)substance and is widely used in a variety of foods. Examples of suchproducts are processed cheese, milk, clotted cream, dairy desserts, icecream mixes, liquid egg, hot-baked flour products, dressings and beer.Nisin is heat-stable and can stand sterilization temperatures withminimal loss of activity. The World Health Organization Committee onBiological Standardization has established an international referencepreparation of nisin, the International Unit (IU hereinafter).DELVOPLUS® and NISAPLIN®, brand names for nisin concentrates aredistributed respectively by DSM and Danisco. DELVOPLUS® and NISAPLIN®contain 2.5% of pure nisin or 1 million IU per gram. Effective levels ofnisin to preserve food products range from 10 to 800 IU/g or 0.25 to 20ppm of nisin.

The invention further relates to a product treated with the anti-fungalcomposition of the invention. The anti-fungal composition of the presentinvention can be used to treat a wide variety of food- and feed productssuch as cheese, shredded cheese, yoghurt, butter, processed meatproducts, sausages, cereals, vegetables, fruits, fruit products andready to use meals. The anti-fungal composition may also be used for thetreatment of beverages such as fruit juices, lemonades, ice-tea, wineand beer. Agricultural applications such as spraying in the field or ingreen houses or post-harvest treatment is also included in thisinvention. Examples of crops are cereals, fruits, vegetables, beans,nuts, herbs, flowers and plants. Also seeds, flower bulbs and seedpotatoes can be treated with the anti-fungal composition of thisinvention. Examples of pharmaceutical or cosmetic applications arecompositions for topical applications, lotions, creams, ointments,shampoos and soaps.

Preferably the natamycin of the invention is incorporated into a foodcoating. Preferably, the coating is used to coat a meat or dairyproduct. All coatings as described in EP 1 239 732B are herewithincorporated by reference in this context. Briefly, such a coating ispreferably used in the coating of a cheese, a sausage or a derivedproduct. All types of polymers described in this patent may also be usedin a coating comprising the natamycin of the invention. Additionally,anti-oxidating agent and/or chelating agent as described in EP 1 239 732B may also be added to the natamycin of the invention and/or to coatingcomprising the natamycin of the invention.

The invention also relates to the use of the anti-fungal composition ofthe invention for the treatment of a product susceptible to fungalspoilage. The invention additionally relates to a method for preservinga product susceptible to fungal spoilage by treating the product withthe anti-fungal composition of the invention.

The present invention is further illustrated by the following examples,which should not be construed as limiting the scope of the invention.

Example 1 Microbiological Method for Determining the Availability of anAnti-Fungal Component

This example describes a microbiological method for determining theavailability of an anti-fungal component within an anti-fungalcomposition. In this example, natamycin is the anti-fungal component.

Filter paper discs (S&S Antibiotics Test Discs no. 321260) with adiameter of 0.6 cm were loaded with the preparation to be tested suchthat each disc was loaded with 40 μg of natamycin, e.g. 50 μl of asample containing 800 ppm of the natamycin to be tested was applied to adisc. The discs were then put on agar, which was seeded with theSaccharomyces cerevisiae ATCC 9763. The petri dishes containing the agarwere then stored for 24 hours at 6° C. to permit the natamycin torelease into the agar. Under these conditions, Saccharomyces does notgrow. As a reference, discs were freshly loaded with a range of knownamounts of natamycin dissolved in aqueous methanol.

The next day, the sample discs were transferred to new petri dishescontaining agar seeded with Saccharomyces cerevisiae. New discs freshlyloaded with a range of known quantities of dissolved natamycin wereprepared for use as a reference. The new dishes with the sample discsand the new reference were stored at 6° C. for 24 hours and the olddishes containing the released natamycin incubated at 30° C. for 24hours.

The size of the inhibition zone is a measure of the natamycin releasedfrom the sample disc. The amount of released natamycin can be calculatedby methods know per se. By repeating the procedure, the releasednatamycin can be measured on a daily basis. Other release time periodsmay alternatively be chosen.

Example 2 Production Process of Natamycin with Improved Properties

A fermentation broth of Streptomyces natalensis containing natamycinhaving an average particle diameter of about 10 micrometer was incubatedat a temperature of 35° C. for 3 hours. This thermally treated broth wasfurther treated to a gravity gradient centrifugation. The centrifuge wasoperated under such conditions that most of the biomass solids wasremoved together with the supernatant. This treatment resulted in asuspension of 70 w/w % natamycin (anhydrous basis) on dry matter. Thenatamycin yield was about 97%. A propanol solution was then added toobtain a natamycin suspension containing 35 v/v % propanol. A NaOHsolution was added to increase the pH of the suspension to 10.4 in orderto dissolve the natamycin. Subsequently, the natamycin solution wasfiltered using a membrane filter press equipped with filter cloth havinga pore size of at least 10 μm. Subsequently, the filter cake was washedusing a 35 v/v % propanol solution. After the membrane filtration, adepth filtration was performed using a pore size of 0.25 microns.Subsequently, the depth filtration filters were washed using a 35 v/v %propanol solution. After the depth filtration, the pH of the filtratewas lowered to 6.0 by reducing the pH 0.3 pH units per 5 minutes byadding hydrochloric acid in order to precipitate the natamycin from saidfiltrate. Subsequently, the mother liquor was then removed by membranefiltration. To further reduce the water content, the crystals werewashed using a propanol solution. The produced natamycin cake was driedusing a vacuum dryer. The dried crystals were then micronised to between1 to 10 micrometers.

Example 3 Comparison of the Release Rate of the Natamycin of theInvention with Natamycin of the Prior Art

Using the method described in example 1, the release rate profile of thenew slow release natamycin was compared to the release rate profile ofknown commercial natamycin DELVOCID® (DSM, The Netherlands). Suspensionsof the natamycin preparations were prepared in such a manner that eachsuspension contained 800 ppm natamycin as such. 50 μl of each of therespective mixtures was applied on a paper filter disc and the releaserate was analysed using the method of example 1. The results aresummarized in the following tables.

TABLE 1 Initial release rate (40 μg carrier loading) Release rate afterRelease rate after 24 hours 48 hours (μg/24 hours) (μg/24 hours) Lowrelease natamycin <2 <1.8 DELVOCID ® <3 <2

TABLE 2 Decrease in release rate after 9 days % decrease in release rateLow release natamycin 35 (10 μg carrier loading) Low release natamycin45 (40 μg carrier loading) DELVOCID ® 58 (40 μg carrier loading)

TABLE 3 Release rate after 11 days Release rate (μg/24 hours) Lowrelease natamycin 0.35 (5 μg carrier loading) Low release natamycin 0.6(10 μg carrier loading) Low release natamycin 1.2 (40 μg carrierloading) DELVOCID ® 1.1 (10 μg carrier loading)

The slow release natamycin according to the invention clearly has alower release rate than a commercially available natamycin preparation(DELVOCID®).

Example 4 Comparison of the Release Rate of the Natamycin of theInvention with Natamycin of the Prior Art in Beverages

The release rate of the new slow release natamycin was compared withthat of known commercial natamycin DELVOCID® Instant (DSM, TheNetherlands) by measuring their chemical stability in various beverages.The natamycin preparations were suspended in beverages to a finalconcentration of 150 to 170 ppm natamycin. The natamycin containingbeverages were stored at 4° C. and the natamycin concentration in thebeverage was measured in time. The results are summarized in thefollowing tables.

TABLE 1 Details of beverages Beverage Manufacturer pH Tomato juiceAppelsientje (J44E3 34:38), Friesland Foods, 4.2 The Netherlands Ice teaPlus Supermarket (05125SE25056), The Netherlands 3.2

TABLE 2 Natamycin degradation in beverages Total degradation (ppm) TimeSlow release Natamycin from Beverage (weeks) natamycin DELVOCID ®Instant Difference Tomato juice 1 5.1 8.6 3.5 2 13.1 13.7 0.6 3 20.126.5 6.4 4 23.2 27.9 4.7 5 28.9 32.6 3.7 Ice tea 1 3.4 14.9 11.5 2 5.415.5 11.1 3 3.2 15.3 12.1 4 15.0 28.2 13.2 5 17.4 31.7 14.3

The data show that less slow release natamycin than natamycin inDELVOCID® Instant is degraded in tomato juice and ice tea. Since onlythe dissolved fraction of a natamycin formulation is suspectible todegredation, the results indicate that the dissolved fraction of slowrelease natamycin is smaller than that of natamycin in DELVOCID®Instant. Hence, the release rate of slow release natamycin is lower thanthat of DELVOCID® Instant in both beverages.

1. A natamycin comprising a release rate that decreases by from 1 to 45percent after at least 9 days, when contacted on a carrier with an agarsurface of 0.6 cm diameter and a carrier loading of from 5 to 40 μg ofnatamycin.
 2. A natamycin exhibiting a release rate ranging from 6 to 10percent of the initial carrier loading after at least 9 days, whentested on a carrier with an agar surface of 0.6 cm diameter, and acarrier loading of from 5 to 40 μg of natamycin.
 3. The natamycinaccording to claim 1, further comprising a release rate ranging from 0.1to 2.0 μg per 24 hours over the first 24 hours, when contacted on acarrier with an agar surface of 0.6 cm diameter, and a carrier loadingof 40 μg of natamycin.
 4. The natamycin according to claim 1, furthercomprising a release rate ranging from 0.1 to 1.0 μg per 24 hours overthe first 24 hours, when contacted on a carrier with an agar surface of0.6 cm diameter, and a carrier loading of 10 μg of natamycin.
 5. Thenatamycin according to claim 1, wherein a water-miscible solvent ispresent with the natamycin.
 6. The natamycin according to claim 1,wherein said natamycin is incorporated into a food coating.
 7. Anantifungal composition comprising natamycin according to claim
 1. 8. Theantifungal composition according to claim 7, wherein said antifungalcomposition further comprises at least another antimicrobial agentselected from the group consisting of a weak acid preservative, sulphurdioxide, sulphite, nitrate, nitrite, dimethyl dicarbonate, biphenyl,diphenyl, orthophenylphenol, thiabendazole, an inorganic acid, animidazole, a bacteriocin, any antifungal compound known in the artcapable of being used as a preservative for food products, cropprotection or after-harvest treatment of fruits, vegetables or cereals,pharmaceutical or cosmetic products and lysozyme.
 9. The antifungalcomposition according to claim 8, wherein said bacteriocin is nisin orpediocin.
 10. The antifungal composition according to claim 8, whereinsaid weak acid preservative is selected from the group consisting ofsorbic acid, propionic acid, benzoic acid, a p-hydroxybenzoic acid,lactic acid, citric acid, acetic acid, or an alkali metal or alkaliearth metal salt thereof.
 11. A product selected from the groupconsisting of cheese, sausages or other meat product such as poultry,seafood, and bakery products treated with the natamycin according toclaim
 1. 12. A product treated with the antifungal composition accordingto claim
 7. 13. A product treated with the antifungal compositionaccording to claim
 8. 14. A product according to claim 12, wherein saidproduct is selected from the group consisting of cheese, shreddedcheese, yoghurt, butter, processed meat products, sausages, cereals,vegetables, fruits, fruit products, ready to use meals, beans, nuts,herbs, flowers, plants, seeds, flower bulbs, seed potatoes, andbeverages such as fruit juices, lemonades, ice-tea, wine and beer.
 15. Aproduct according to claim 12, wherein said product is selected from thegroup consisting of compositions for topical applications, lotions,creams ointments, shampoos and soaps.
 16. A method for preserving aproduct susceptible to fungal spoilage by treating the product with theantifungal composition according to claim 7.